Salmonella must endure intervals of acid stress in widely diverse environments ranging from pond water to fused phagolysosomes in infected macrophages. We have discovered a unique survival response that effectively protects the cells from extreme low pH. This acid tolerance response (ATR) is composed of two distinct stages. A pre-acid shock stage triggered below pH 6 and a post-acid shock stage induced below pH 4.5. Both stages are required for the cell to survive low pH (pH 3.3) These findings present a challenging biological question. How does a neutralophillic organism protect itself from severe acid stress? Preliminary results indicate preshock produces an inducible pH homeostasis system that augments the housekeeping system as external pH decreases below pH 4 while acid shock appears to generate a set of proteins designed to minimize acid damage to macromolecules. An adapted culture in which both systems are engaged can survive pH 3.3 stress approximately 1000 fold better than unadapted cultures. Biochemical and genetic approaches have revealed integral roles for the Mg+2 dependent proton-translocating ATPase (atp) and the iron-regulatory locus, fur, in the ATR system. This proposal has three aims designed to investigate the ATR phenomenon at the biochemical, genetic and molecular levels. They are to (1) define the genetic components of the ATR system, (2) reveal the protective mechanisms used and (3) assign specific functions to specific atr loci. Genetic characterization will occur through the generation of acid-sensitive atr insertions (including lacZ operon fusion) and the identification of new regulatory loci. These genes will be cloned and the most intriguing subjected to molecular analysis. As part of this aim, the Salmonella fur product will be scrutinized for properties commensurate to its role in acid tolerance. We will examine the preshock inducible pH homeostasis mechanism by measuring buffering capacity, proton influx and proton efflux in adapted and unadapted cells. Insights regarding post shock protection of cellular macromolecules will be gained by using acid-sensitive indicator proteins and observing acid-damaged DNA repair. The unifying theme throughout this project is to correlate specific atr mutations with the loss of a given repair or protection system. This proposal presents a comprehensive research plan designed to reveal the elegant molecular response of salmonella to low pH. On a higher level, the knowledge gained will address fundamental questions concerning general defense strategies used by micro-organisms to survive harsh environments.